Saa domain-specific antibodies and peptide antagonists and use thereof to treat inflammatory diseases

ABSTRACT

Isolated SAA peptides, fusion proteins and compositions comprising such are provided as are domain-specific SAA antibodies. Methods of treating sepsis and endotoxemia are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/846,749, filed Jul. 16, 2013, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparentheses. Full citations for these references may be found at the endof the specification immediately preceding the claims. The disclosuresof these publications, and of all books, patents and patent applicationpublications referred to herein, are hereby incorporated by reference intheir entireties into the subject application to more fully describe theart to which the subject application pertains.

Bacterial infection and sepsis are the most common causes of death inthe intensive care unit, annually claiming >225,000 victims in the U.S.alone. The pathogenesis of sepsis remains poorly understood, but isattributable to dysregulated systemic inflammation partly mediated bymacrophages/monocytes (1,2). Macrophages/monocytes are equipped withpattern recognition receptors [PRRs, such as the Toll-like receptors(TLRs), TLR2, TLR4, and TLR9] (3-5), and can bind variouspathogen-associated molecular patterns (PAMPs, such as bacterialpeptidoglycan, endotoxin, and CpG-DNA) (6-9). Consequently, these innateimmune cells release a wide array of early proinflammatory cytokinessuch as TNF, IL-1 and IFN-γ 10-13. Excessive release of early cytokinescontributes to the pathogenesis of LSI 12, 14-16. However, thetherapeutic windows for these early mediators are relatively narrow(FIG. 1), prompting the search for other “late” proinflammatorymediators that may offer better therapeutic opportunities.

A decade ago, this laboratory made the seminal finding that highmobility group box-1 (HMGB1) was released from macrophages or monocytesin response to exogenous PAMPs (e.g., endotoxin or CpG-DNA) (17,18) orendogenous cytokines (e.g., TNF or IFN-γ) (17,19). Upon binding to thereceptor for advanced glycation end products (RAGE), TLR2 or TLR4(20-22), HMGB 1 induces the expression of various cytokines, chemokines,and adhesion molecules (20,21,23-29). Consequently, extracellular HMGB1functions as an alarmin signal to alert, recruit and activate innateimmune cells (30-34), thereby sustaining rigorous and potentiallyinjurious LSI. During endotoxemia or sepsis (induced by cecal ligationand puncture, CLP), circulating HMGB1 increased to plateau levelsbetween 24-36 h (FIG. 1) (17,35). This late appearance precedes theonset of animal lethality, and distinguishes HMGB1 from TNF and otherearly cytokines (36). The pathogenic role of HMGB1 was inferred from theobservations that HMGB1-neutralizing antibodies (17,35,37) andinhibitors (e.g., tanshinones, ethyl pyruvate, nicotine, stearoyllysophosphatidylcholine, epigallocatechin-3-gallate, nicotine, choline,GTS-21, and spermine) (17,38,39,39-47) confer protection against lethalendotoxemia and sepsis, even when the first dose of antidote was given24 h after CLP—a time point when mice had developed clear signs ofsepsis, including lethargy, diarrhea, and piloerection. Conversely,administration of exogenous HMGB1 to mice recapitulated many clinicalmanifestations of sepsis, including fever 48, derangement of intestinalbarrier function 49, and tissue injury (50-53). Collectively, these dataestablish HMGB1 as a critical “late” mediator of sepsis with a widertherapeutic window (36, 54-56) (FIG. 1).

On one hand, early cytokines TNF and IFN-γ can directly stimulatemacrophages or monocytes to release HMGB1 (17,19), thereby contributingto LPS-induced HMGB1 release. On the other hand, these early cytokinesalso alter the expression of liver-derived APPs, which may thenparticipate in the regulation of HMGB1 release. For instance, TNF, IL-1,IL-6 57,58 and IFN-γ (58) inhibited the hepatic expression of a negativeAPP, fetuin-A, which functions as a negative regulator of HMGB 1 releaseduring cerebral ischemia (59), endotoxemia and sepsis (58). However, thepossible roles of other positive APPs in the regulation of HMGB1 releasehave not been identified.

In 1976, serum amyloid A (SAA) was first isolated from human serum as a12 kDa protein (60) that shared identical N-terminal amino acid sequencewith the previously characterized 8.5 kDa tissue amyloid A (AA) protein.Subsequently, it was found that exogenous endotoxin (61,62) orendogenous cytokines (e.g., TNF, IL-1β and IFN-γ) (63-67) can all induceSAA expression in both hepatocytes and extrahepatic cells, such asmacrophages/monocytes (68), endothelial cells, smooth muscle cells,adipocytes (69), intestinal epithelial cells (70), and neurons (71,72).Consequently, circulating SAA levels are dramatically elevated (up to1000-folds) within 16-24 h of endotoxemia as a result of the de novoexpression of early cytokine inducers and the subsequent synthesis ofSAAs (61,73,74).

Clinically, SAA levels have been regarded as a hallmark/risk factor ofmany diseases including atherosclerosis (75), cardiovascular diseases(76,77), Crohn's disease, ulcerative colitis (78), as well as LSIdiseases (such as endotoxemia and sepsis) (79-81). Upon secretion,extracellular SAA acts as a chemoattractant for inflammatory cells suchas macrophages/monocytes (82-84), T cells (85) and mast cells (86).Furthermore, it activates innate immune cells (e.g., macrophages,monocytes, neutrophils and mast cells) to produce various cytokines andchemokines (e.g., TNF, IL-1β, IL-6, IL-10, GM-CSF, IL-8, MCP-1, MIP-1α,and MIP-3α) (87-94). Finally, SAA stimulates non-immune cells to releaseother proinflammatory factors such as the group II secretoryphospholipase A2 (sPLA2, in smooth muscle cells) (95,96) andprostaglandins (in endothelial cells) (97). However, it is not knownwhether SAA occupies an important role in the regulation of HMGB1release, thereby functioning as an “intermediate” (as opposed to TNFbeing “early” and HMGB1 being “late”) mediators of LSI (FIG. 1).

Extensive studies have revealed distinct functional domains in SAA thatare specifically responsible for: 1) SAA secretion (i.e., the signalsequence, D1); 2) HDL binding (D2); 3) cellular adhesion (D3); 4)as-yet-undefined function (D4); 5) protease cleavage (between residues76-77); and 6) cell activation (D5) (FIG. 2). For instance, the signalsequence directs pro-SAA to the endoplasmic reticulum, and is cleavedprior to extracellular secretion of the mature SAA (98). The α-helicalD2 domain serves as the driving force for binding to high densitylipoprotein (HDL) (99,100). Within the D2 domain, the first 10-15residues are particularly critical for the amyloidosis of SAA (101),because deletion or mutation in this region impaired amyloid fibrilformation (102,103). The D3 domain contains YIGSD laminin-related andRGN fibronectin-related motifs, and can inhibit T cell and plateletadhesion to extracellular matrixes (104,105). At position 76-77 lies acleavage site for the leukocyte-derived proteases, which break theserine-leucine bond (106-108) to liberate the 8.5-kDa amyloid A (AA) and3.5-kDa (D5) fragments. The accumulation of the AA precipitates theformation of amorphous amyloid fibril deposits—amyloidosis in peripheraltissues with progressive loss of organ function. On the other hand, the3.5-kDa fragment may be proinflammatory, because a synthetic peptidecorresponding to residues 98-104 stimulates human CD4 T cells to produceIFN-γ (109).

The present invention addresses the need for improved therapies, basedon SAA, to combat sepsis and endotoxemia.

SUMMARY OF THE INVENTION

An isolated peptide of 6 to 20 consecutive amino acids is providedcomprising (i) 6 to 18 consecutive amino acids of SEQ ID NO:1, (ii) 6 to18 consecutive amino acids of SEQ ID NO:2, (iii) 6 to 18 consecutiveamino acids of SEQ ID NO:3, or (iv) 6 to 18 consecutive amino acids ofSEQ ID NO:4.

Also provided is a composition comprising an isolated peptide asdescribed herein and a carrier.

Also provided is a fusion protein comprising an isolated peptide asdescribed herein bonded via a peptide bond at an N-terminal thereof or aC-terminal thereof to a second peptide, polypeptide or protein. Anisolated cDNA which encodes a fusion protein as described herein isprovided.

Also provided is an isolated antibody directed to a domain of SAA,wherein the antibody cross-reacts with isolated peptide P8 (SEQ ID NO:2) and/or with isolated peptide P4 (SEQ ID NO:3), or an antigen-bindingfragment of such antibody.

An isolated antibody is provided directed to a domain of human SAA,wherein the antibody cross-reacts with isolated peptide P8 (SEQ ID NO:2)or with isolated peptide P4 (SEQ ID NO:3), or with one of isolatedpeptides SEQ ID NOS:14-18, or an antigen-binding fragment of suchantibody.

A composition is provided comprising an isolated antibody as describedherein or antigen-binding fragment of such antibody as described herein,and a carrier.

A hybridoma which produces an isolated antibody as described herein isprovided. An isolated cDNA which encodes an isolated antibody asdescribed herein is provided.

A method is provided of treating or preventing sepsis or endotoxemia ina subject, comprising administering to the subject an amount of anisolated peptide as described herein, the fusion protein as describedherein, an antibody or fragment thereof as described herein, or acomposition as described herein, effective to treat or prevent sepsis orendotoxemia.

A method is provided of treating or preventing an inflammatory conditionin a subject, comprising administering to the subject an amount of anisolated peptide as described herein, the fusion protein as describedherein, an antibody or fragment thereof as described herein, or acomposition as described herein, effective to treat or prevent aninflammatory condition.

Also provided is a peptide having a biological activity of a peptidecomprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ IDNO:9, but not comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8 or SEQ ID NO:9, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. “Early” versus “late” mediators of lethal systemic inflammation(LSI).

FIG. 2. Functional domains of SAA.

FIG. 3A-3D. Identification of SAA as an anti-HMGB1 IgG-cross-reactingprotein in septic patients. A, Western blots of serum proteins. Notethat anti-HMGB1 IgGs recognized both the 30 kDa HMGB 1 and a 12 kDaprotein (termed “P12”) in septic patients. B, Mass spectrometry analysisof P12. C, D, Immunoassays of total SAAs and multiple other cytokines byELISA and Cytokine Antibody Arrays. Note that serum HMGB1 levelspositively correlated with SAAs and several sepsis surrogate markers.

FIG. 4A-4C. Human SAA, but not SAA1, induced HMGB1 release in vitro. A,Amino acid sequence of human and murine SAAs. B, Western blots of humanSAA and SAA1. Note that the anti-HMGB1 IgGs paradoxically cross-reactedwith huSAA, but not huSAA1. C, HuSAA, but not huSAA1, induced HMGB1release. Murine macrophages and human monocytes were stimulated withhuSAA or huSAA1 for 16 h, and extracellular HMGB 1 levels weredetermined by Western blots.

FIG. 5A-5C. Epitope mapping of SAA-specific antibodies using a peptidelibrary. A, Sequence of synthetic SAA peptides. B, Effects of anti-SAAIgGs on SAA-induced HMGB1 release. Macrophages were stimulated with SAAin the absence or presence of different (R1, R2, R3) IgGs, and HMGB1release was assayed by Western blotting. C, Epitope mapping by dot blot.Note that all rabbit IgGs capable of protecting against lethalendotoxemia cross-reacted with P8 peptide.

FIG. 6A-6C. Divergent effects of SAA and SAA-neutralizing IgGs on LSI.A, SAA exacerbated LPS-induced animal lethality. Balb/C mice were givenLPS (5 mg/kg, i.p.) either alone or in combination with SAA (0.8 mg/kg)to determine their effects on animal survival rates. B, SAA-specificIgGs protected mice against lethal endotoxemia. Balb/C mice were givenLPS (15 mg/kg) in combination with either control IgGs (50 mg/kg, R2) orSAA-reacting IgGs (R1 and R3, 50 mg/kg). C, peptide-specific anti-SAAIgGs rescued mice from lethal sepsis. Balb/C mice were subjected tosepsis by CLP, and irrelevant (“control”, 50 mg/kg) or SAApeptide-specific IgGs (50 mg/kg) were given at +6, +24, and +48 h postCLP. Shown in the figure is a summary of two independent experimentswith similar results.

FIG. 7. SAA peptide (P4) inhibited SAA-induced HMGB 1 release. P4peptide corresponded to residues 1-18 that are critical for HDL bindingand amyloidosis.

FIG. 8. Effects of SAA peptides on septic animal survival. Variouspeptides (6-mers) corresponding to the P8 peptide (18-mer) of the humanSAA (“HuSAA”) or murine SAA1 (“MuSAA1”) protein were synthesized(AnaSpec Inc.), and tested for efficacy in animal model of CLP-inducedsepsis. At +20 and +48 h post CLP, mice were administered with eithersaline (i.p., 0.2 ml/mouse) or each peptide (30 mg/kg), and animalsurvival rates were monitored for two weeks. Shown in the figure is asummary of 2 independent experiments with similar results.

FIG. 9. Four fusion proteins were constructed that each displayed 3 ofthe 12 peptides (10-mer, sequences shown) on the surface of insolubleand highly immunogenic molecules. These fusion proteins were expressed,purified, and used to immunize mice to generate hybridomas usingstandard procedures. Hybridoma cell culture supernatants were screenedfor cross-reactivity with human or murine SAAs, and ability to inhibitSAA-induced release of nitric oxide or G-CSF in macrophage cultures.From top to bottom, sequences are SEQ ID NOS: 18, 19, 20, 13, 14, 21,16, 17, 22, 23, 15 and 24.

DETAILED DESCRIPTION OF THE INVENTION

An isolated peptide of 6 to 20 consecutive amino acids is providedcomprising (i) 6 to 18 consecutive amino acids of SEQ ID NO:1, (ii) 6 to18 consecutive amino acids of SEQ ID NO:2, (iii) 6 to 18 consecutiveamino acids of SEQ ID NO:3, or (iv) 6 to 18 consecutive amino acids ofSEQ ID NO:4.

This invention also provides a peptide fragment of SAA which inhibitsSAA-induced HMGB1 release in a mammal. This invention also provides apeptide fragment of SAA which inhibits sepsis mortality rates in apopulation of septic mammals. This invention also provides a peptidefragment of SAA which treats sepsis mortality in a mammal.

The following peptides, and peptides comprising such, are provided:

P8 human SAA- SEQ ID NO: 1 REANYIGSDKYFHARGNY P8 mouse SAA- SEQ ID NO: 2KEANWKNSDKYFHARGNY P4 human SAA- SEQ ID NO: 3 RSFFSFLGEAFDGARDMWSEQ ID NO: 4 GFFSFVHEAFQGAGDMWR P8-8- SEQ ID NO: 5 NSDKYF P8-7-SEQ ID NO: 6 NWKNSD P8-6- SEQ ID NO: 7 KEANWK P8-4- SEQ ID NO: 8 KYFHARP8-5- SEQ ID NO: 9 HARGNY P8-1- SEQ ID NO: 10 REANYI P8-2- SEQ ID NO: 11NYIGSD P8-3- SEQ ID NO: 12 GSDKYF.

The fragments provided are isolated peptides not having the naturallyoccurring sequence of full length serum amyloid A. Moreover, given thedifferent domain functions of serum amyloid A, as described herein, thepeptides comprising less than all the domains described clearly havedifferent characteristics to the naturally-occurring SAA protein (a 12KDa protein).

In an embodiment, the isolated peptide comprises SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In an embodiment, theisolated peptide comprises SEQ ID NO:5. In an embodiment of the isolatedpeptide, all the amino acid residues of the peptide are D-amino acids.In an embodiment of the isolated peptide, all the amino acid residues ofthe peptide are L-amino acids.

The 6 to 20 consecutive amino acid isolated peptide can be any one of20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids inlength. Each individual peptide length recited herein is encompassedwithin the invention as an individual embodiment. In addition, each ofall the length ranges within the recited lengths is also encompassedwithin the invention as an individual embodiment. For example, thisinvention encompasses the isolated peptides of 15-20 amino acids inlength, the isolated peptides of 14-20 amino acids in length, theisolated peptides of 14-19 amino acids in length, the isolated peptidesof 13-14 amino acids in length and so forth.

In an embodiment, the isolated peptide does not consist of SEQ ID NO:10.In an embodiment, the isolated peptide does not consist of SEQ ID NO:11.In an embodiment, the isolated peptide does not consist of SEQ ID NO:12.

In an embodiment, the isolated peptide comprises SEQ ID NO:5, 6, 7, 8 or9, and has a sequence as set forth in any of the sequences above exceptfor comprising one or more amino acid substitutions in the isolatedpeptide that are not in the SEQ ID NO:5, 6, 7, 8 or 9 portion thereof.The isolated peptide may have one of: 80% or greater identity with anyone of SEQ ID NO:5, 6, 7, 8 or 9, 85% or greater identity with any oneof SEQ ID NO:5, 6, 7, 8 or 9, 90% or greater identity with any one ofSEQ ID NO:5, 6, 7, 8 or 9, 95% or greater identity with any one of SEQID NO:5, 6, 7, 8 or 9, or 99% identity with any one of SEQ ID NO:5, 6,7, 8 or 9.

The substitution variants of the invention have at least one amino acidresidue in the isolated peptide removed and a different residue insertedin its place. The sites of greatest interest for substitutionalmutagenesis are outside the core sequences, i.e. in residues other thanSEQ ID:5, 6, 7, 8 or 9. In an embodiment, one or more of thesubstitutions is a conservative substitution. Conservative substitutionsare shown in Table 1 under the heading of “conservative substitutions.”In an embodiment, one or more of the substitutions is a substitution asset forth in the third column of Table 1.

TABLE 1 Amino Acid Substitutions Original Residue ConservativeSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Modifications in the biological properties of the isolated peptide areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gln;(3) Acidic (negatively charged): Asp, Glu;(4) Basic (positively charged): Lys, Arg;(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

In an embodiment, the substitution variants of the invention comprisenon-conservative substitutions. Non-conservative substitutions are madeby exchanging a member of one of the classes (1) through (6) for anotherclass.

In an embodiment, the substitution or substitutions improve solubilityof the isolated peptide in the serum of a subject. In an embodiment, thesubstitution or substitutions improve the half-life of the isolatedpeptide in the body of a subject. An improvement is relative to thecorresponding non-substituted peptide.

This invention also provides any of the above-described peptides withone substitution or with two substitutions in the sequence SEQ ID NO:5,6, 7, 8 or 9.

The isolated peptide can comprise both D-amino acids and L-amino acids.In an embodiment, all the amino acid residues of the peptide are D-aminoacids. In an embodiment, all the amino acid residues of the peptide areL-amino acids.

Also provided is a fusion protein comprising an isolated peptide asdescribed herein bonded via a peptide bond at an N-terminal thereof or aC-terminal thereof to a second peptide, polypeptide or protein. A fusionprotein is provided comprising the isolated peptide as describedhereinabove, joined at an N-terminal amino acid or C-terminal amino acidthereof by a peptide bond to a second peptide or polypeptide or protein.In an embodiment, the fusion protein comprising the isolated peptide hasa longer half-life in a human subject than the isolated peptide alonedoes. In an embodiment, the fusion protein comprising the isolatedpeptide is more soluble in the serum of a human subject than theisolated peptide alone is. In a preferred embodiment, the fusion proteinis an isolated recombinant fusion protein created by recombinant DNAtechnology. In an embodiment, the peptide is fused to a functionaldomain of a second peptide or polypeptide or protein. In an embodiment,the peptide is fused to a functional domain of a second peptide which isa cell-penetrating peptide. In an embodiment, the cell-penetratingpeptide is TAT, transportan or penetratin. In an embodiment, the peptideis fused to an immunoglobulin constant domain (Fc).

In an embodiment, the peptide is fused to a human immunoglobulinconstant domain, i.e. a polypeptide having a sequence identical to ahuman immunoglobulin constant domain but not obtained from an actualhuman subject. In an embodiment, the isolated peptide is bonded via apeptide bond at an N-terminal thereof or a C-terminal thereof to aimmunoglobulin fragment crystallizable region (Fc). In an embodiment,the Fc has a sequence identical to a human Fc.

In an embodiment, the fusion protein comprising the isolated peptide hasa longer half-life in a human subject than the isolated peptide alonedoes. In an embodiment, the fusion protein comprising the isolatedpeptide is more soluble in the serum of a human subject than theisolated peptide alone is.

In an embodiment, “isolated” as used herein means not naturallyoccurring without the hand of man.

Also provided is a composition comprising an isolated peptide asdescribed herein and a carrier. Also provided is a compositioncomprising a fusion protein as described herein and a carrier. Theinvention encompasses compositions comprising the isolated peptidesdescribed herein or the fusion proteins described herein. In anembodiment, the composition is a pharmaceutical composition. In anembodiment the composition or pharmaceutical composition comprising oneor more of the isolated peptides described herein or the fusion proteinsdescribed herein is substantially pure with regard to the isolatedpeptides described herein or the fusion proteins described herein. Acomposition or pharmaceutical composition comprising one or more of theisolated peptides described herein or the fusion proteins describedherein is “substantially pure” with regard to that when at least 60% ofa sample of the composition or pharmaceutical composition exhibits asingle species of the isolated peptide or fusion protein. Asubstantially pure composition or pharmaceutical composition comprisingone or more of the isolated peptides described herein or the fusionproteins described herein can comprise, in the portion thereof which isthe isolated peptide or fusion protein, 60%, 70%, 80% or 90% of theisolated peptide or fusion protein of the single species, more usuallyabout 95%, and preferably over 99%. Purity or homogeneity may tested bya number of means well known in the art, such as polyacrylamide gelelectrophoresis or HPLC.

In an embodiment, the composition is a dimer or trimer of the isolatedpeptides. In an embodiment, the composition is a dimer or trimer of thefusion proteins.

Compositions or pharmaceutical compositions disclosed herein preferablycomprise stabilizers to prevent loss of activity or structural integrityof the peptide or fusion protein due to the effects of denaturation,oxidation or aggregation over a period of time during storage andtransportation prior to use. The compositions or pharmaceuticalcompositions can comprise one or more of any combination of salts,surfactants, pH and tonicity agents such as sugars can contribute toovercoming aggregation problems. Where a composition or pharmaceuticalcomposition of the present invention is used as an injection, it isdesirable to have a pH value in an approximately neutral pH range, it isalso advantageous to minimize surfactant levels to avoid bubbles in theformulation which are detrimental for injection into subjects. In anembodiment, the composition or pharmaceutical composition is in liquidform and stably supports high concentrations of bioactive antibody insolution and is suitable for parenteral administration, includingintravenous, intramuscular, intraperitoneal, intradermal and/orsubcutaneous injection. In an embodiment, the composition orpharmaceutical composition is in liquid form and has minimized risk ofbubble formation and anaphylactoid side effects. In an embodiment, thecomposition or pharmaceutical composition is isotonic. In an embodiment,the composition or pharmaceutical composition has a pH of 6.8 to 7.4.

In an embodiment the isolated peptides or fusion proteins disclosedherein are lyophilized and/or freeze dried and are reconstituted foruse.

The invention encompasses compositions comprising the isolated peptidesor fusion proteins described herein in a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” includesany material (including mixtures) which, when combined with an activeingredient, allows the ingredient to retain biological activity and isnon-reactive with the subject's immune system. Examples include, but arenot limited to, any of the standard pharmaceutical carriers such as oneor more of phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 20th Ed. Mack Publishing, 2000). Innon-limiting examples, the can comprise one or more of dibasic sodiumphosphate, potassium chloride, monobasic potassium phosphate,polysorbate 80 (e.g.2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl(E)-octadec-9-enoate),disodium edetate dehydrate, sucrose, monobasic sodium phosphatemonohydrate, and dibasic sodium phosphate dihydrate.

The compositions or pharmaceutical compositions described herein canalso be lyophilized or provided in any suitable forms including, but notlimited to, injectable solutions or inhalable solutions, gel forms andtablet forms.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997).

In some embodiments, therapeutic administration of the isolated peptide,or of the composition comprising such, advantageously results in reducedincidence and/or amelioration of one or more symptoms of the sepsis orendotoxemia condition. In an embodiment, the said composition is apharmaceutical composition. In some embodiments, therapeuticadministration of the isolated fusion-protein or the compositioncomprising such advantageously results in reduced incidence and/oramelioration of one or more symptoms of the sepsis or endotoxemiacondition. In an embodiment, the said composition is a pharmaceuticalcomposition.

With respect to the therapeutic methods described herein, reference tocompositions includes compositions comprising one or more additionalagents, unless otherwise indicated. These compositions may furthercomprise suitable excipients, such as pharmaceutically acceptableexcipients including buffers, which are well known in the art. Thepresent invention can be used alone or in combination with other methodsof treatment. In an embodiment, the other method of treatment comprisean anti-inflammation therapy and/or an anti-bacterial therapy.

The isolated peptides, fusion proteins, or compositions comprising such,can be administered to an subject via any suitable route. It should beapparent to a person skilled in the art that the examples describedherein are not intended to be limiting but to be illustrative of thetechniques available. Accordingly, in some embodiments, the they areadministered to a subject in accord with known methods, such asintravenous administration, e.g., as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerebrospinal, transdermal, subcutaneous, intra-articular,sublingually, intrasynovial, via insufflation, intrathecal, oral,inhalation or topical routes. Administration can be systemic, e.g.,intravenous administration, or localized. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution.

In some embodiments, the isolated peptide, fusion protein, orcomposition comprising such, is administered via site-specific ortargeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources or local delivery catheters, such as infusion catheters,indwelling catheters, or needle catheters, synthetic grafts, adventitialwraps, shunts and stents or other implantable devices, site specificcarriers, direct injection, or direct application. See, e.g., PCTPublication No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Various formulations of the isolated peptide or fusion protein of theinvention may be used for administration. In some embodiments, theisolated peptide or fusion protein of the invention may be administeredneat. In some embodiments, the isolated peptide or fusion protein of theinvention and a pharmaceutically acceptable excipient may be in variousformulations. Pharmaceutically acceptable excipients are known in theart, and are relatively inert substances that facilitate administrationof a pharmacologically effective substance. For example, an excipientcan give form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000.

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular subject andthat subject's medical history.

Therapeutic formulations of the peptide or fusion protein used inaccordance with the present invention are prepared for storage by mixingwith optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000), in the form of lyophilized formulations oraqueous solutions. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andmay comprise buffers such as phosphate, citrate, and other organicacids; salts such as sodium chloride; antioxidants including ascorbicacid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride,benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens,such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Liposomes containing the peptide or fusion protein are prepared bymethods known in the art, such as described in Epstein, et al., Proc.Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad.Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the isolated peptide or fusion protein,which matrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms. The tablets or pills of thenovel composition can be coated or otherwise compounded to provide adosage form affording the advantage of prolonged action. For example,the tablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer that serves toresist disintegration in the stomach and permits the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

Emulsion compositions of the invention can be those prepared by mixingan isolated peptide or fusion protein of the invention with Intralipid™or the components thereof (soybean oil, egg phospholipids, glycerol andwater).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulized by use of gases. Nebulized solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Also provided is an isolated antibody directed to a domain of SAA,wherein the antibody cross-reacts with isolated peptide P8 (SEQ ID NO:2) and/or with isolated peptide P4 (SEQ ID NO:3), or an antigen-bindingfragment of such antibody.

Also provided is an isolated antibody directed to a domain of human SAA,wherein the antibody cross-reacts with isolated peptide P8 (SEQ ID NO:2)or with isolated peptide P4 (SEQ ID NO:3), or with one of isolatedpeptides SEQ ID NOS: 13-17, or an antigen-binding fragment of suchantibody.

In an embodiment, the isolated antibody reacts with one of amino acidsequences ANYQNADQYF (SEQ ID NO:15), DKYFHARGNY (SEQ ID NO:14), orFRPEGLPEKY (SEQ ID NO:17). In an embodiment, the isolated antibodyreacts with amino acid sequence KNPNHFRPEG (SEQ ID NO:16).

In an embodiment, the isolated antibody is non-naturally occurring inthat it does not exist absent the hand of man in its production.

In an embodiment, the isolated antibody inhibits SAA-induced nitricoxide production when applied to a human macrophage culture. In anembodiment, the isolated antibody inhibits SAA-induced G-CSF productionwhen applied to a human macrophage culture.

In a preferred embodiment, the antibody cross-reacts with isolatedpeptide P8 (SEQ ID NO: 2). In a preferred embodiment, the antibody doesnot bind an epitope bound by monoclonal antibody mc29 and/or wherein theantibody does not bind an epitope bound by monoclonal antibody mc4.

In an embodiment, the antibody is a monoclonal antibody. In anembodiment, the antibody is a chimeric antibody, humanized antibody orhuman antibody.

A composition is provided comprising an isolated antibody as describedherein or antigen-binding fragment of such antibody as described herein,and a carrier.

As used herein, the term “antibody” refers to an intact antibody, i.e.with complete Fc and Fv regions. “Fragment” refers to any portion of anantibody, or portions of an antibody linked together, such as, innon-limiting examples, a Fab, F(ab)₂, a single-chain Fv (scFv), which isless than the whole antibody but which is an antigen-binding portion andwhich competes with the intact antibody, of which it is a fragment, forspecific binding. As such, a fragment can be prepared, for example, bycleaving an intact antibody or by recombinant means. See generally,Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989), hereby incorporated by reference in its entirety).Antigen-binding fragments may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies or bymolecular biology techniques. In some embodiments, a fragment is an Fab,Fab′, F(ab′)₂, F_(d), F_(v), complementarity determining region (CDR)fragment, single-chain antibody (scFv), (a variable domain light chain(V_(L)) and a variable domain heavy chain (V_(H)) linked via a peptidelinker. In an embodiment the linker of the scFv is 10-25 amino acids inlength. In an embodiment the peptide linker comprises glycine, serineand/or threonine residues. For example, see Bird et al., Science, 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-5883 (1988) each of which are hereby incorporated by referencein their entirety), or a polypeptide that contains at least a portion ofan antibody that is sufficient to confer SAA domain-specific antigenbinding on the polypeptide, including a diabody. From N-terminus toC-terminus, both the mature light and heavy chain variable domainscomprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia& Lesk, J. Mol. Biol. 196:901-917 (1987), or Chothia et al., Nature342:878-883 (1989), each of which are hereby incorporated by referencein their entirety). As used herein, the term “polypeptide” encompassesnative or artificial proteins, protein fragments and polypeptide analogsof a protein sequence. A polypeptide may be monomeric or polymeric. Asused herein, an F_(d) fragment means an antibody fragment that consistsof the V_(H) and CH1 domains; an F_(v) fragment consists of the V₁ andV_(H) domains of a single arm of an antibody; and a dAb fragment (Wardet al., Nature 341:544-546 (1989) hereby incorporated by reference inits entirety) consists of a V_(H) domain. In some embodiments, fragmentsare at least 5, 6, 8 or 10 amino acids long. In other embodiments, thefragments are at least 14, at least 20, at least 50, or at least 70, 80,90, 100, 150 or 200 amino acids long.

The term “monoclonal antibody” as used herein refers to an antibodymember of a population of substantially homogeneous antibodies, i.e.,the individual antibodies comprising the population are identical exceptfor possible mutations, e.g., naturally occurring mutations, that may bepresent in minor amounts. Thus, the modifier “monoclonal” indicates thecharacter of the antibody as not being a mixture of discrete antibodies.In certain embodiments, such a monoclonal antibody typically includes anantibody comprising a polypeptide sequence that binds a target SAAdomain, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones, or recombinant DNA clones. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins. Thus an identified monoclonal antibody can beproduced by non-hybridoma techniques, e.g. by appropriate recombinantmeans once the sequence thereof is identified.

In an embodiment of the inventions described herein, the antibody isisolated. As used herein, in an embodiment, the term “isolated antibody”refers to an antibody that by virtue of its origin or source ofderivation has one, two, three or four of the following: (1) is notassociated with naturally associated components that accompany it in itsnative state, (2) is free of other proteins from the same species, (3)is expressed by a cell from a different species, and (4) does notnaturally occur absent the hand of man.

In an embodiment the composition or pharmaceutical compositioncomprising one or more of the antibodies or fragments described hereinis substantially pure with regard to the antibody or fragment. Acomposition or pharmaceutical composition comprising one or more of theantibodies or fragments described herein is “substantially pure” withregard to the antibody or fragment when at least about 60 to 75% of asample of the composition or pharmaceutical composition exhibits asingle species of the antibody or fragment. A substantially purecomposition or pharmaceutical composition comprising one or more of theantibodies or fragments described herein can comprise, in the portionthereof which is the antibody or fragment, 60%, 70%, 80% or 90% of theantibody or fragment of the single species, more usually about 95%, andpreferably over 99%. Antibody purity or homogeneity may tested by anumber of means well known in the art, such as polyacrylamide gelelectrophoresis or HPLC.

As used herein, a “human antibody” unless otherwise indicated is onewhose sequences correspond to (i.e. are identical in sequence to) anantibody that could be produced by a human and/or has been made usingany of the techniques for making human antibodies as disclosed herein,but not one which has been made in a human. This definition of a humanantibody specifically excludes a humanized antibody. A “human antibody”as used herein can be produced using various techniques known in theart, including phage-display libraries (e.g. Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991),hereby incorporated by reference in its entirety), by methods describedin Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,p. 77 (1985) (hereby incorporated by reference in its entirety); Boerneret al., J. Immunol., 147(1):86-95 (1991) (hereby incorporated byreference in its entirety), van Dijk and van de Winkel, Curr. Opin.Pharmacol., 5: 368-74 (2001) (hereby incorporated by reference in itsentirety), and by administering the antigen to a transgenic animal thathas been modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;6,150,584 and 6,162,963 to Kucherlapati et al. regarding XENOMOUSE™technology, each of which patents are hereby incorporated by referencein their entirety), e.g. Veloclmmune® (Regeneron, Tarrytown, N.Y.), e.g.UltiMab® platform (Medarex, now Bristol Myers Squibb, Princeton, N.J.).See also, for example, Li et al., Proc. Natl. Acad. Sci. USA,103:3557-3562 (2006) regarding human antibodies generated via a humanB-cell hybridoma technology. See also KM Mouse® system, described in PCTPublication WO 02/43478 by Ishida et al., in which the mouse carries ahuman heavy chain transchromosome and a human light chain transgene, andthe TC mouse system, described in Tomizuka et al. (2000) Proc. Natl.Acad. Sci. USA 97:722-727, in which the mouse carries both a human heavychain transchromosome and a human light chain transchromosome, both ofwhich are hereby incorporated by reference in their entirety. In each ofthese systems, the transgenes and/or transchromosomes carried by themice comprise human immunoglobulin variable and constant regionsequences.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are sequences of human origin or identical thereto other thanantibodies naturally occurring in a human or made in a human.Furthermore, if the antibody (e.g. an intact antibody rather than, forexample, an Fab fragment) contains a constant region, the constantregion also is derived from such human sequences, e.g., human germlinesequences, or mutated versions of human germline sequences. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences. In one non-limiting embodiment, where the humanantibodies are human monoclonal antibodies, such antibodies can beproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

In an embodiment, the anti-SAA antibody described herein is arecombinant human antibody. The term “recombinant human antibody”, asused herein, includes all human antibodies that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic or transchromosomalfor human immunoglobulin genes or a hybridoma prepared therefrom,antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, antibodies isolated from arecombinant, combinatorial human antibody library, and antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of all or a portion of a human immunoglobulin gene, sequencesto other DNA sequences. Such recombinant human antibodies have variableregions in which the framework and CDR regions are derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region (HVR) of the recipient are replaced by residuesfrom a HVR of a non-human species (donor antibody) such as mouse, rat,rabbit, or nonhuman primate having the desired specificity, affinity,and/or capacity. In some instances, FR residues of the humanimmunoglobulin variable domain are replaced by corresponding non-humanresidues. These modifications may be made to further refine antibodyperformance. Furthermore, in a specific embodiment, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. In an embodiment, the humanized antibodies do notcomprise residues that are not found in the recipient antibody or in thedonor antibody. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct.Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma &Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433(1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409, the contents of eachof which references and patents are hereby incorporated by reference intheir entirety. In one embodiment where the humanized antibodies docomprise residues that are not found in the recipient antibody or in thedonor antibody, the Fc regions of the antibodies are modified asdescribed in WO 99/58572, the content of which is hereby incorporated byreference in its entirety.

Techniques to humanize a monoclonal antibody are described in U.S. Pat.Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; and 6,180,370, the content of each of which ishereby incorporated by reference in its entirety.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including antibodies having rodent or modified rodent V regions andtheir associated complementarity determining regions (CDRs) fused tohuman constant domains. See, for example, Winter et al. Nature 349:293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA 86: 4220-4224(1989), Shaw et al. J. Immunol. 138: 4534-4538 (1987), and Brown et al.Cancer Res. 47: 3577-3583 (1987), the content of each of which is herebyincorporated by reference in its entirety. Other references describerodent hypervariable regions or CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant domain. See, for example, Riechmann et al. Nature 332: 323-327(1988), Verhoeyen et al. Science 239: 1534-1536 (1988), and Jones et al.Nature 321: 522-525 (1986), the content of each of which is herebyincorporated by reference in its entirety. Another reference describesrodent CDRs supported by recombinantly veneered rodent frameworkregions—European Patent Publication No. 0519596 (incorporated byreference in its entirety). These “humanized” molecules are designed tominimize unwanted immunological response toward rodent anti-humanantibody molecules which limits the duration and effectiveness oftherapeutic applications of those moieties in human recipients. Theantibody constant region can be engineered such that it isimmunologically inert (e.g., does not trigger complement lysis). See,e.g. PCT Publication No. WO99/58572; UK Patent Application No.9809951.8. Other methods of humanizing antibodies that may also beutilized are disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867;5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO01/27160 (each incorporated by reference in their entirety).

Other forms of humanized antibodies have one or more CDRs (CDR L1, CDRL2, CDR L3, CDR H1, CDR H2, or CDR H3) which are altered with respect tothe original antibody, which are also termed one or more CDRs “derivedfrom” one or more CDRs from the original antibody.

In embodiments, the antibodies or fragments herein can be producedrecombinantly, for example antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial human antibody library, antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes.

In an embodiment, the anti-SAA antibody described herein is capable ofspecifically binding or specifically binds an SAA domain. As usedherein, the terms “is capable of specifically binding” or “specificallybinds” refers to the property of an antibody or fragment of binding tothe (specified) antigen with a dissociation constant that is <1 μM,preferably <1 nM and most preferably <10 pM. In an embodiment, the Kd ofthe antibody (or fragment) for SAA is 250-500 pM. An epitope that“specifically binds” to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecular entityis said to exhibit “specific binding” or “preferential binding” if itreacts or associates more frequently, more rapidly, with greaterduration and/or with greater affinity with a particular cell orsubstance than it does with alternative cells or substances. An antibody“specifically binds” or “preferentially binds” to a target if it bindswith greater affinity, avidity, more readily, and/or with greaterduration than it binds to other substances. It is also understood byreading this definition that, for example, an antibody (or moiety orepitope) that specifically or preferentially binds to a first target mayor may not specifically or preferentially bind to a second target. Assuch, “specific binding” or “preferential binding” does not necessarilyrequire (although it can include, in an embodiment) exclusive binding.

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding portion thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. The antibody or fragment can be, e.g., any of an IgG, IgD, IgE,IgA or IgM antibody or fragment thereof, respectively. In an embodimentthe antibody is an immunoglobulin G. In an embodiment the antibodyfragment is a fragment of an immunoglobulin G. In an embodiment theantibody is an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4. In an embodimentthe antibody comprises sequences from a human IgG1, human IgG2, humanIgG2a, human IgG2b, human IgG3 or human IgG4. A combination of any ofthese antibodies subtypes can also be used. One consideration inselecting the type of antibody to be used is the desired serum half-lifeof the antibody. For example, an IgG generally has a serum half-life of23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days. (Abbas A K,Lichtman A H, Pober J S. Cellular and Molecular Immunology, 4th edition,W.B. Saunders Co., Philadelphia, 2000, hereby incorporated by referencein its entirety).

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “V_(H).” Thevariable domain of the light chain may be referred to as “V_(L).” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites. The term “variable” refers to the fact thatcertain portions of the variable domains differ extensively in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. However, the variabilityis not evenly distributed throughout the variable domains of antibodies.It is concentrated in three segments called hypervariable regions (HVRs)both in the light-chain and the heavy-chain variable domains. The morehighly conserved portions of variable domains are called the frameworkregions (FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (4 based on the amino acid sequences of theirconstant domains.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “hypervariable region” or “HVR” when used herein refers to theregions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the V_(H) (H1, H2, H3) and three in theV_(L) (L1, L2, L3). In native antibodies, H3 and L3 display the mostdiversity of the six HVRs, and H3 in particular is believed to play aunique role in conferring fine specificity to antibodies. See, e.g., Xuet al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods inMolecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).Indeed, naturally occurring camelid antibodies consisting of a heavychain only are functional and stable in the absence of light chain. See,e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff etal., Nature Struct. Biol. 3:733-736 (1996). A number of HVR delineationsare in use and are encompassed herein. The Kabat ComplementarityDetermining Regions (CDRs) are based on sequence variability and are themost commonly used (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991) hereby incorporated by reference in its entirety).Chothia refers instead to the location of the structural loops (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent acompromise between the Kabat HVRs and Chothia structural loops, and areused by Oxford Molecular's AbM antibody modeling software. The “contact”HVRs are based on an analysis of the available complex crystalstructures. HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35(H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH.The variable domain residues are numbered according to Kabat et al.,supra, for each of these definitions.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine of the Fc region may be removed, for example, duringproduction or purification of the antibody, or by recombinantlyengineering the nucleic acid encoding a heavy chain of the antibody.Accordingly, an intact antibody as used herein may be an antibody withor without the otherwise C-terminal cysteine.

Compositions or pharmaceutical compositions comprising the antibodies,ScFvs or fragments of antibodies disclosed herein are preferablycomprise stabilizers to prevent loss of activity or structural integrityof the protein due to the effects of denaturation, oxidation oraggregation over a period of time during storage and transportationprior to use. The compositions or pharmaceutical compositions cancomprise one or more of any combination of salts, surfactants, pH andtonicity agents such as sugars can contribute to overcoming aggregationproblems. Where a composition or pharmaceutical composition of thepresent invention is used as an injection, it is desirable to have a pHvalue in an approximately neutral pH range, it is also advantageous tominimize surfactant levels to avoid bubbles in the formulation which aredetrimental for injection into subjects. In an embodiment, thecomposition or pharmaceutical composition is in liquid form and stablysupports high concentrations of bioactive antibody in solution and issuitable for parenteral administration, including intravenous,intramuscular, intraperitoneal, intradermal and/or subcutaneousinjection. In an embodiment, the composition or pharmaceuticalcomposition is in liquid form and has minimized risk of bubble formationand anaphylactoid side effects. In an embodiment, the composition orpharmaceutical composition is isotonic. In an embodiment, thecomposition or pharmaceutical composition has a pH or 6.8 to 7.4.

In an embodiment the ScFvs or fragments of antibodies disclosed hereinare lyophilized and/or freeze dried and are reconstituted for use.

Examples of pharmaceutically acceptable carriers include, but are notlimited to, phosphate buffered saline solution, sterile water (includingwater for injection USP), emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline, for example 0.9% sodium chloride solution, USP. Compositionscomprising such carriers are formulated by well known conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; andRemington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000, the content of each of which is hereby incorporated inits entirety). In non-limiting examples, the can comprise one or more ofdibasic sodium phosphate, potassium chloride, monobasic potassiumphosphate, polysorbate 80 (e.g.2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl(E)-octadec-9-enoate),disodium edetate dehydrate, sucrose, monobasic sodium phosphatemonohydrate, and dibasic sodium phosphate dihydrate.

The antibodies, or fragments of antibodies, or compositions, orpharmaceutical compositions described herein can also be lyophilized orprovided in any suitable forms including, but not limited to, injectablesolutions or inhalable solutions, gel forms and tablet forms.

A hybridoma is provided which produces an antibody as described herein.

An isolated cDNA is provided which encodes an antibody as describedherein.

An isolated cDNA is provided which encodes a fusion protein as describedherein.

The methods of routes of administration described hereinabove withregard to peptides are also applicable to the antibodies, antibodyfragments, and compositions comprising either as described herein.

A method is provided of treating or preventing sepsis or endotoxemia ina subject, comprising administering to the subject an amount of anisolated peptide as described herein, the fusion protein as describedherein, an antibody or fragment thereof as described herein, or acomposition as described herein, effective to treat or prevent sepsis orendotoxemia.

A method is provided of treating or preventing an inflammatory conditionin a subject, comprising administering to the subject an amount of anisolated peptide as described herein, the fusion protein as describedherein, an antibody or fragment thereof as described herein, or acomposition as described herein, effective to treat or prevent aninflammatory condition.

In an embodiment, the methods further comprise administering an amountof exogenous HDL to the subject sufficient to reduce plasma levels ofSAA in a subject.

In an embodiment of the methods, the antibody or fragment, or thecomposition comprising such, is administered. In an embodiment, theisolated peptide, or the fusion protein or composition comprising such,is administered.

In an embodiment of the methods herein, the isolated peptide, fusionprotein, antibody or composition is administered to the subject prior tothe onset of sepsis or endotoxemia, for example, to a subject at riskfor sepsis. In an embodiment of the methods herein, the isolatedpeptide, fusion protein, antibody or composition is administered to thesubject after the onset of sepsis or endotoxemia or inflammatorycondition, for example, within 2 hrs., 5 hrs., 10 hrs., 24 hrs. or 48hrs. or more after the onset of sepsis or endotoxemia or inflammatorycondition. In an embodiment of the methods herein, the isolated peptide,fusion protein, antibody or composition is administered to the subjectshowing sepsis symptoms or showing endotoxemia symptoms or exhibitingthe inflammatory condition.

Also provided is a peptide having a biological activity of a peptidecomprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ IDNO:9, but not comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8 or SEQ ID NO:9, respectively.

In an embodiment of any of the peptides described herein, the peptideattenuates SAA-induced HMGB1 release from a cell, or in a subject. In anembodiment of any of the antibodies or fragments described herein, theantibody or fragment attenuates SAA-induced HMGB1 release from a cell,or in a subject.

The term “subject” is intended to include mammals, e.g., humans, dogs,cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and alsoincludes avians. In a preferred embodiment of the invention, the subjectis a human.

“And/or” as used herein, for example, with option A and/or option B,encompasses the separate embodiments of (i) option A, (ii) option B, and(iii) option A plus option B.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Introduction

Despite recent advances in antibiotic therapy and intensive care,bacterial infection and sepsis remain widespread problems in criticallyill patients. The high mortality of sepsis is partly mediated bybacterial toxins, which stimulate macrophages/monocytes to sequentiallyrelease early (e.g., TNF and IFN-γ) and late (e.g., HMGB1 and histones)proinflammatory mediators. Anti-TNF agents can be protective in animalmodels of endotoxemic shock if given prophylactically; whereas agentscapable of inhibiting HMGB1 release or activities rescue mice fromlethal sepsis even when these anti-HMGB1 agents are first given 24 hafter disease onset. To search for other HMGB1 regulators, wesystematically monitored the dynamic changes of the circulating levelsof HMGB1 and multiple other cytokines/chemokines in septic patients byWestern blots and Antibody Arrays. Intriguingly, a positive APP, humanserum amyloid A (SAA), but not SAA1, cross-reacted with some rabbitanti-HMGB1 antibodies, and surprisingly induced HMGB1 release in murinemacrophage and human monocyte cultures. Furthermore, recombinant SAAprotein exacerbated endotoxin-mediated animal lethality; whereas SAAdomain-specific neutralizing antibodies and peptide antagonistsconferred protection against lethal endotoxemia and sepsis.

Results

Discovery of human SAA, but not SAA1, as an HMGB1 inducer. To search forpotential “intermediate” mediators that could contribute to HMGB1release, the kinetic changes of serum levels of HMGB1 along withmultiple other cytokines in a group of septic patients admitted to theNSUH were characterized subsequent to the approval by the institutionalIRB ethics committee. In 8 out of 23 septic patients, serum HMGB1 levelspositively correlated with the clinical scores—circulating HMGB1 levelsreturned to baselines when these patients recovered from the illness(110). In a subset of septic patients, some anti-HMGB1 IgGsparadoxically cross-reacted with a 12 kDa protein (denoted as “P12”)(FIG. 3A), which was identified as a member of the human SAA family bymass spectrometry analysis (FIG. 3B). Furthermore, serum HMGB1 levelsappeared to positively correlate with the serum levels of total SAAs (asmeasured by ELISA, FIG. 3C), several sepsis surrogate markers (e.g.,IL-6, GRO/KC, IL-8, MCP-1, and RANTES), as well as a cytoprotectivepeptide, EGF (41, 111-114) (FIG. 3D). However, most of theseinflammatory mediators (e.g., IL-6, IL-8, EGF and KC) were unable toinduce HMGB1 release in vitro (data not shown), eliminating theirpotential involvement in the regulation of HMGB 1 release during sepsis.

Overall, there is a >95-98% amino acid sequence homology between membersof the human or murine SAA families, and a >75% identity across humanand murine families (FIG. 4A), making it seemingly difficult todistinguish between different SAA members by immunoassays. Indeed, thepolyclonal antibodies raised against human SAA cross-reacted with bothhuman SAA and SAA1 on Western blots (FIG. 4B, bottom panel).Intriguingly, our rabbit anti-HMGB1 IgGs “specifically” recognized humanSAA, but not SAA1, on Western blots (FIG. 4B), implicating a possibilityof selectively immunodetecting human SAA with appropriate antibodies.Despite the overwhelming sequence identity between human SAA and SAA1(FIG. 4A), their capacities in stimulating HMGB1 release weresurprisingly distinct. At physiologically relevant concentrations (1-10μg/ml), human SAA (Cat. No. 300-13, PeproTech) effectively induced HMGB1release in both murine macrophage and human monocyte cultures (FIG. 4C).In a sharp contrast, human SAA1 (Cat. No. 300-53, PeproTech) barelytriggered HMGB1 release when given at essentially comparableconcentrations (FIG. 4C).

Epitope mapping of SAA-neutralizing antibodies. To test the possibilitythat antibodies or peptides antagonists targeting different SAA domainsmay divergently affect the outcomes of LSI, SAA-specific antibodies weregenerated, and the epitopes of these polyclonal IgGs determined using apeptide library (18-mer offset by 6) spanning the entire SAA protein(FIG. 5A). Consistent with recent reports that SAA autoantibodies weredetected in both healthy individuals and patients with variousautoimmune diseases (115,116), it was found that all rabbits producesmall amounts of anti-SAA autoantibodies (immunoreactive to human SAAand, fortunately, not to murine SAA) prior to SAA immunization.Following repetitive immunizations, the titers of anti-SAA IgGs weredramatically elevated in most rabbits (e.g., R1 and R3). Notably, someSAA-specific antibodies significantly attenuated SAA-induced HMGB1release in vitro (FIG. 5B), whereas the anti-SAA-IgG-mediated inhibitionwas impaired by pre-incubation with the P8 peptide (FIG. 5B).Interestingly, these SAA-neutralizing antibodies all cross-reacted withthe P8 peptide (FIG. 5C), suggest the importance of domain specificneutralizing antibodies to inhibit SAA-induced HMGB1 release.

Anti-SAA antibodies protected mice against lethal endotoxemia andsepsis. To understand the role of SAA in lethal systemic inflammatorydiseases, the effect of SAA supplementation or inhibition on animalsurvival was examined in animal models of lethal endotoxemia and sepsis.Recombinant SAA protein dramatically exacerbated LPS-induced animallethality (FIG. 6A), whereas repetitive administration ofSAA-neutralizing IgGs (at +0.5 and +24 h after LPS) promoted asignificant protection against lethal endotoxemia (FIG. 6B). Moreover,polyclonal IgGs targeting specific SAA domains (e.g., the P7/P8peptides) conferred significant protection in a clinically relevantanimal model of CLP-induced sepsis (FIG. 6C), suggesting that dampeningundesired SAA functions may be beneficial during LSI. Since “early”(such as TNF) and possibly even “intermediate” (such as SAA) cytokinesmay still be required for the early innate immunity against infection,the abolition of the SAA-mediated early host defense might still bedetrimental (117). Indeed, combinational administration of two MAbs(mc29 and mc4; 1:1), given (50 mg/kg) prophylactically at 12 h before,and additionally (33 mg/kg) at 12 h after, CLP, adversely reduced animalsurvival rates from 60% to 20% (118).

To gain insight into the mechanism of SAA action, the capacities ofvarious SAA peptides was also explored in their effect on SAA-inducedHMGB1 release. Consistent with a previous report that a peptidecorresponding to residues 98-104 induced IFN-γ production in T cells(109), it was found that a peptide corresponding to residues 85-102(i.e., the “P18” peptide; 50 μg/ml) elevated SAA (100 ng/ml)-inducedHMGB1 release by 2-3 folds. In a sharp contrast, several peptidescorresponding to residues 1-18 (the “P4” peptide, FIG. 7) or 25-42 (the“P8” peptide, data not shown) dose-dependently inhibited SAA-inducedHMGB 1 release, suggesting that selective SAA domain-specific peptidesmay be developed as antagonists to block SAA-mediated HMGB1 release.

To explore the therapeutic potential of SAA peptides, a peptide librarywas synthesized that spanned a smaller region of SAA. Notably, severalsmaller peptides specifically corresponding to the murine SAA1 (but nothuman SAA) P8 peptide antagonist significantly rescued mice from lethalsepsis even when the first dose of peptides were given at 20 h post theonset of sepsis (FIG. 8).

Generation and screening SAA peptide-reacting monoclonal antibodies:four fusion proteins were constructed that each displayed of the 12peptides shown in FIG. 9. (10-mer sequences shown) on the surface ofinsoluble and highly immunogenic molecules. These fusion proteins wereexpressed, purified, and used to immunize mice to generate hybridomasusing standard procedures. Hybridoma cell culture supernatants werescreened for cross-reactivity with human or murine SAAs, and ability toinhibit SAA-induced release of nitric oxide or G-CSF in macrophagecultures. The data obtained is summarized in the Table 2.

TABLE 2 Characterization of mouse anti-SAA peptide hybridomas. SAA-neutralizing Activity Clone Peptide Reactivity ↓ Nitric ↓ G- # NameSequence HuSAAs MuSAAs Oxide CSF 8 Pm8-4 KNSDKYFHAR − ++ 75% − (SEQ IDNO: 13) 9 Pm8-5 DKYFHARGNY + − (SEQ ID NO: 14) 20 Ph8-3 ANYQNADQYF + ++77% + (SEQ ID NO: 15) 22 Ph8-3 ANYQNADQYF + + (SEQ ID NO: 15) 30 Pm8-5DKYFHARGNY ++ − 89% ++ (SEQ ID NO: 14) 31 Ph18-2 KNPNHFRPEG ++ + 81%(SEQ ID NO: 16) 32 Ph18-2 KNPNHFRPEG ++ +++ (SEQ ID NO: 16) 34 Ph18-2KNPNHFRPEG + + 84% − (SEQ ID NO: 16) 35 Ph18-3 FRPEGLPEKY +++ ++ (SEQ IDNO: 17) 36 Ph18-3 FRPEGLPEKY ++ + (SEQ ID NO: 17) 38 Ph18-3 FRPEGLPEKY ++++ 81% ++ (SEQ ID NO: 17) 39 Ph18-3 FRPEGLPEKY + − (SEQ ID NO: 17)

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1. An isolated antibody directed to a domain of human SAA, wherein theantibody cross-reacts with isolated peptide P8 (SEQ ID NO:2) or withisolated peptide P4 (SEQ ID NO:3), or with one of isolated peptides SEQID NOS:14-18, or an antigen-binding fragment of such antibody.
 2. Theisolated antibody or fragment of claim 1, wherein the antibodycross-reacts with isolated peptide P8 (SEQ ID NO: 2).
 3. (canceled) 4.The isolated antibody of claim 1, which reacts with the amino acidsequence ANYQNADQYF (SEQ ID NO:15), DKYFHARGNY (SEQ ID NO:14), orFRPEGLPEKY (SEQ ID NO:18).
 5. The isolated antibody of claim 1, whichreacts with the amino acid sequence KNPNHFRPEG (SEQ ID NO:17).
 6. Theisolated antibody or fragment of claim 1, wherein the antibody is achimeric antibody, humanized antibody or human antibody.
 7. The isolatedantibody or fragment of claim 1, wherein the antibody is a monoclonalantibody.
 8. A composition comprising the isolated antibody of claim 1,or antigen-binding fragment of such antibody, and a carrier.
 9. Thecomposition of claim 8, wherein the carrier is a pharmaceuticallyacceptable carrier.
 10. A method of treating or preventing sepsis orendotoxemia in a subject, comprising administering to the subject anamount of an isolated antibody or fragment thereof of claim 1, effectiveto treat or prevent sepsis or endotoxemia.
 11. A method of treating orpreventing an inflammatory condition in a subject, comprisingadministering to the subject an amount of an isolated antibody orfragment thereof of claim 1, effective to treat or prevent aninflammatory condition.
 12. The method of claim 10, further comprisingadministering an amount of exogenous HDL to the subject sufficient toreduce plasma levels of SAA in a subject. 13-14. (canceled)
 15. Anisolated non-naturally occurring peptide of 6 to 20 consecutive aminoacids comprising (i) 6 to 18 consecutive amino acids of SEQ ID NO:1,(ii) 6 to 18 consecutive amino acids of SEQ ID NO:2, (iii) 6 to 18consecutive amino acids of SEQ ID NO:3, or (iv) 6 to 18 consecutiveamino acids of SEQ ID NO:4. 16-21. (canceled)
 22. A fusion proteincomprising the isolated peptide of claim 15 bonded via a peptide bond atan N-terminal thereof or a C-terminal thereof to a second peptide,polypeptide or protein.
 23. The fusion protein of claim 22, wherein theisolated peptide is bonded via a peptide bond at an N-terminal thereofor a C-terminal thereof to a immunoglobulin fragment crystallizableregion (Fc).
 24. (canceled)
 25. The fusion protein of claim 22, whereinthe fusion protein comprising the isolated peptide has a longerhalf-life in a human subject than the isolated peptide alone does. 26.The fusion protein of claim 22, wherein the fusion protein comprisingthe isolated peptide is more soluble in the serum of a human subjectthan the isolated peptide alone is. 27-29. (canceled)
 30. A method oftreating or preventing sepsis or endotoxemia or an inflammatorycondition in a subject, comprising administering to the subject anamount of a fusion protein of claim 22, effective to treat or preventsepsis or endotoxemia.
 31. (canceled)
 37. A hybridoma which produces anantibody of claim
 1. 38. An isolated cDNA encoding an antibody ofclaim
 1. 39. An isolated cDNA encoding a fusion protein of claim 22.